Tumors develop in hosts endowed with a highly complex immune system that includes various lymphocyte subsets capable of recognizing and destroying transformed cells. It is now widely accepted that, while lymphocytes may constantly patrol tumor formation, cancer cells develop molecular strategies to evade immune surveillance, which are competitively selected under the pressure of the host immune system. This dynamic process, termed “cancer immunoediting”, is thought to constitute a major obstacle to cancer immunotherapy.
Among multiple immune evasion mechanisms, it was recently shown that leukemia and lymphoma primary samples often down-regulate the non-classical MHC (major histocompatibility complex) protein, ULBP1, which is critical for recognition of hematological tumors by γδ T-cells expressing the counter-receptor NKG2D (Lanca, T. et al., “The MHC class Ib protein ULBP1 is a nonredundant determinant of leukemia/lymphoma susceptibility to gammadelta T-cell cytotoxicity”, Blood 115:2407-2411; 2010).
γδ T-cells are innate-like lymphocytes that account for 1-10% of peripheral blood lymphocytes (PBL) of healthy individuals and are capable of targeting a significant fraction of hematological tumor cell lines tested in the laboratory. However, it was demonstrated that many lymphoid leukemia primary samples are resistant to fully-activated Vγ9Vδ2 T-cells (Lanca, T. et al., “The MHC class Ib protein ULBP1 is a nonredundant determinant of leukemia/lymphoma susceptibility to gammadelta T-cell cytotoxicity”, Blood 115:2407-2411, 2010; Gomes, A. Q. et al., “Identification of a panel of ten cell surface protein antigens associated with immunotargeting of leukemias and lymphomas by peripheral blood gammadelta T cells”, Haematologica 95:1397-1404, 2010), the dominant subset of γδ PBLs. Furthermore, clinical trials involving the in vivo administration of activators of Vγ9Vδ2 T-cells have shown limited success, with objective responses restricted to 10-33% of patients with either hematological or solid tumors. Even more modest has been the outcome of trials involving the adoptive transfer of activated/expanded Vδ2+ cells, since no objective responses have been reported. In fact, the simple ex vivo expansion of autologous Vδ2+ T-cells, whose surveillance the tumor managed to escape in vivo, may be condemned to little therapeutic effect upon re-injection into the patient.
Cancer immunotherapy relies on tumor cell recognition by cytotoxic lymphocytes. γδ T-cells are a population of MHC-unrestricted killer lymphocytes that play critical roles in various animal tumor models. This notwithstanding, it was showed that a large proportion of human hematological tumors is resistant to γδ peripheral blood lymphocytes (PBLs) activated with specific agonists to the highly prevalent Vγ9Vδ2 T-cell receptor (TCR). This likely constitutes an important limitation to current γδ-T-cell-mediated immunotherapy.
Therefore, it is critical to invest in strategies that endow γδ T-cells with additional recognition machinery to detect tumors that have resisted the natural components present in vivo.
Natural cytotoxicity receptors were identified by A. Moretta and co-workers over a decade ago, and were shown to play critical synergistic roles in the anti-tumor functions of Natural Killer (NK) cells. In fact, NKp30 and NKp46 are widely considered to be two of the most specific NK cell markers.
The document written by von Lilienfeld-Toal, M., J. Nattermann, et al. (2006). “Activated gammadelta T cells express the natural cytotoxicity receptor natural killer p 44 and show cytotoxic activity against myeloma cells.” Clin Exp Immunol 144(3): 528-533. discloses the expression of a NK receptor on peripheral blood γδ T lymphocytes. However, disclosed γδ T cells are different from those stated in the present invention. The document also reveals, contrary to the present invention, that NKp30 is not expressed in such γδ T cells. Some of differences are:                The treatment (IFN-γ, TNF-α+anti-CD3 monoclonal antibody+IL-1β+IL-2+IL-15);        The phenotype of γδ T cells (NKp30 and NKp46 moreover, 62% of γδ T cells belong to the Vδ2+ subtype;        Less than 20% of the γδ T cells are CD56+;        Less than 20% of the γδ T cells are CD8+;        The expression levels of NKp44 are not modulated by stimulation of the γδTCR/CD3 complex (thus, different molecular mechanisms are involved that induce NKp44 expression on those cells)        
This document describes peripheral blood γδ T cells expressing NKp44. NKp44 was functional and was involved in the recognition and elimination of tumor cells (myeloma cells). However, only 8±7% of γδ T cells expressed NKp44 and most (62%) of γδ T cells belonged to the Vδ2+ subtype (they were Vδ1−). NKp30 and NKp46 were not expressed by γδ T cells, and less than 20% of these cells were CD8+ or CD56+.
Thus, the γδ T cell line described is totally different from our identified γδ T cell line: typically more than 95% of γδ T cells in our cell line belong to the Vδ1+ subtype and a high percentage of these Vδ1+ γδ T cells (typically more than 30%) express functional NKp44. Importantly, NKp44 demonstrate synergism with NKp30 to greatly enhance the ability to recognize and kill tumor cells (FIG. 5B). This cooperation between NCR is critical to obtain the desired effect (elimination of cancer disease), and is absent in the previously identified NKp44+ γδ T cells.
The document written by Rey, J., C. Veuillen, et al. (2009). “Natural killer and gammadelta T cells in haematological malignancies: enhancing the immune effectors.” Trends Mol Med 15(6): 275-284. discloses the common expression of the NKG2D receptor on NK cells and on γδ T cells. This document also states that the expansion and activation of γδ T cells can generate anti-tumor responses. However, contrary to the present invention the γδ cells are NKp30− and NKp46−.
The document WO 00/44893 (Palmetto Richland Memorial Hos) discloses a method of treatment of leukemia based on the administration of substantially purified γδ T lymphocytes. The method comprises the preparation of lymphocyte activation and expansion thereof. The main differences are:                The treatment (plate-bound immobilized anti-TCR antibodies+irradiated “feeder” tumor B-cells);        /The γδ T cells (NKp30−, NKp44−, NKp46−, CD8−).        
The document WO 2011/053750 (Emory University) discloses a method of reducing cancer in a patient, comprising the steps of isolating a population of cytotoxic cells, like γδ T cells; administration of the therapeutic agent and the cytotoxic cells. The main difference to present invention is that γδ T cells are genetically modified to resist chemotherapeutic agents.